To further define the conditions for forming spectrin-hemoglobin cross-linking in human erythrocyte membranes and to examine its possible effects on membrane function, we incubated normal human erythrocytes for up to 3 h in concentrations of H202, varying from 45 to 180 ;.M, in an azide phosphate buffer, pH 7.4. The chemical changes observed indicated that methemoglobin formation occurred early and at a low concentration (45 MM). Morphologic changes characterized by increased echinocyte formation occurred in a dose-dependent fashion. In addition, decreased cell deformability commensurate with increased membrane rigidity was found. Finally, an increase in cell recognition as determined by monocyte phagocytosis and adherence in vitro, as well as decreased phosphatidylcholine accessibility to bee venom phospholipase A2, was found in H202-treated erythrocytes compared with controls. Both of these latter changes were closely correlated with the extent of spectrin-hemoglobin cross-linking.In addition to these protein-mediated interactions, lipid peroxidation also occurred after H202 exposure, as shown by generation of fluorescent amino propene derivatives. The addition of the antioxidant, butylated hydroxytoluene, decreased the fluorescent derivatives, but did not prevent the effects on membrane function. This suggests that lipid peroxidation, though present, was not necessary for the membrane changes found. In contrast, spectrin-hemoglobin aggregation and the alterations in membrane function were completely prevented by prior exposure of the erythrocytes to carbon monoxide.

Direct macrophage recognition of an externalized phosphatidylserine signal on senescent erythrocytes is a process of erythrophagocytic clearance that is in line with the general clearance process of all other circulating cells that become apoptotic. Advances in deciphering this process suggest that oxidation of the erythrocyte's hemoglobin, the salient target of the free radicals encountered in the circulatory environment, may drive subsequent steps. The progressive accumulation of oxidized hemoglobin covalently bound to the membrane skeleton not only disrupts membrane organization but also threatens eventual phospholipid oxidation via a calcium-promoted quasi-lipoxygenase activity. The emergence on the cell surface of a threshold concentration of oxidized phospholipids, principally phosphatidylserine, signals recognition by the CD36 macrophage receptor.

Medicine, law, and social values are not static. Reexamining the ethical tenets of medicine and their application in new circumstances is a necessary exercise. The sixth edition of the American College of Physicians (ACP) Ethics Manual covers emerging issues in medical ethics and revisits older ones that are still very pertinent. It reflects on many of the ethical tensions in medicine and attempts to shed light on how existing principles extend to emerging concerns. In addition, by reiterating ethical principles that have provided guidance in resolving past ethical problems, the Manual may help physicians avert future problems. The Manual is not a substitute for the experience and integrity of individual physicians, but it may serve as a reminder of the shared duties of the medical profession.

The optional Escherichia coli prr locus restricts phage T4 mutants lacking polynucleotide kinase or RNA ligase. Underlying this restriction is the specific manifestation of the T4-induced anticodon nuclease, an enzyme which triggers the cleavage-ligation of the host tRNALYS. We report here the molecular cloning, nucleotide sequence and mutational analysis of prr-associated DNA. The results indicate that prr encodes a latent form of anticodon nuclease consisting of a core enzyme and cognate masking agents. They suggest that the T4-encoded factors of anticodon nuclease counteract the prr-encoded masking agents, thus activating the latent enzyme. The encoding of a tRNA cleavage-ligation pathway by two separate genetic systems which cohabitate E.coli may provide a clue to the evolution of RNA splicing mechanisms mediated by proteins.

Dehydrated hereditary stomatocytosis (DHSt) is an autosomal dominant congenital hemolytic anemia with moderate splenomegaly and often compensated hemolysis. Affected red cells are characterized by a nonspecific cation leak of the red cell membrane, reflected in elevated sodium content, decreased potassium content, elevated MCHC and MCV, and decreased osmotic fragility. The majority of symptomatic DHSt cases reported to date have been associated with gain-of-function mutations in the mechanosensitive cation channel gene, PIEZO1. A recent study has identified two families with DHSt associated with a single mutation in the KCNN4 gene encoding the Gardos channel (KCa3.1), the erythroid Ca 21 -sensitive K 1 channel of intermediate conductance, also expressed in many other cell types. We present here, in the second report of DHSt associated with KCNN4 mutations, two previously undiagnosed DHSt families. Family NA exhibited the same de novo missense mutation as that recently described, suggesting a hot spot codon for DHSt mutations. Family WO carried a novel, inherited missense mutation in the ion transport domain of the channel. The patients' mild hemolytic anemia did not improve post-splenectomy, but splenectomy led to no serious thromboembolic events. We further characterized the expression of KCNN4 in the mutated patients and during erythroid differentiation of CD341 cells and K562 cells. We also analyzed KCNN4 expression during mouse embryonic development.